Screening And Identification Of Differentially Expressed Roteins In Tobacco Leaves Under Low-nitrogen Stress

In order to study the differentially expressed proteins of tobacco leafunder low nitrogen conditions, The protein expression profiles of tobacco leavesharvested at70days after transplanting were analyzed by2-dimensionalelectrophoresis, and five differentially expressed proteins including a putative protein wereidentified. Except for the MCM protein-like protein, the other four differentiallyexpressed proteins of cyclophilin-like protein(CyP2), vacuolar invertase INV2,MAR-binding protein and the one putative protein showed increasedexpression in the low-N nutrition group. To further validate the differential proteinexpression in tobacco leaves under low N, The mRNA expression of the five differentiallyexpressed proteins between low-N and normal-N nutrition were analyzed. The mRNA levelsof the five identified proteins in low-N leaf compared with that in normal-N leaf were in linewith their protein level indicating the same responses were stimulated at both mRNA andprotein level by the low nitrogen treatment.Among the five differentially expressed protein, the cyclophilin-like protein(CyP2),which is suggested to be an unspecific stress-responsive protein, may play an importantrole in the growth process of tobacco under low nitrogen．The mRNA expression level ofcyclophilin gene was analyzed by real—time quantitative PCR. The mRNA expression levelof cyclophilin gene in low nitrogen leaf was increased compared with the mRNA level innormal nitrogen leaf．Gene expression was induced continuously in low nitrogen leaf. Theinduced expression of cyclophilin gene by low nitrogen was confirmed by hydroponicsexperiments under control condition in low nitrogen leaf which was increased comparedwith the mRNA level in normal nitrogen leaf．Cyclophilins is a multi-gene family, in order to further study the function of cyclophilinfamily genes in the tobacco during growth and development, the expression patterns of threecyclophilin genes of tobacco (CyP1, CyP2, CyP40) were analyzed. Results showed indifferent developmental stages, the expression level of CyP40was up-regulated in laterstages (after transplanting60d), CyP1was up-regulated after transplanting40d,50d,60d,CyP2were up-regulated after transplanting30d,40d and60d which suggested the expression of CyP1, CyP2, CyP40were increased in the middle leaves and lower leaves.The expression of Cyclophilin genes in different tissues were increased in the middle leafand lower leaf. CyP1and CyP40with the higher expression in tobacco roots, CyP40alsowas expressed higher in tobacco flowers. CyP2was mainly expressed in tobacco leaves.The results of this paper showed CyP2gene and protein in leaves were induced underlow nitrogen stress condition and is the mainly expressed gene which induced in mature leaf.CyP2as a stress protein might be involve in the tobacco physiological process to responsethe low nitrogen stress and might participate in regulating tobacco leaf growth.